1. Field of the Invention (Technical Field):
The present invention relates to metallopeptides, metal ion-complexed peptidomimetics, and metallo-constructs, including metallopeptide combinatorial libraries, metal ion-complexed peptidomimetic and peptide-like combinatorial libraries and metallo-construct combinatorial libraries, specific for melanocortin receptors, including methods for the use and making of the same. The invention also relates to methods for synthesizing and assembling such libraries, and methods for identification and characterization of library constituents which are capable of binding a melanocortin receptor of interest, or mediating a melanocortin receptor-related biological activity of interest.
2. Background Art
Not that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications ar not to be considered as prior art vis-a-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.
Melanocortin Receptors. A family of melanocortin receptor types and subtypes has been identified, including melanocortin-1 receptors (MC1-R) expressed on normal human melanocytes and melanoma cells, melanocortin-2 receptors (MC2-R) for ACTH (adrenocorticotropin) expressed in cells of the adrenal gland, melanocortin-3 and melanocortin-4 receptors (MC3-R and MC4-R) expressed primarily in cells in the hypothalamus, mid-brain and brainstem, and melanocortin-5 receptors (MC5-R), expressed in a wide distribution of peripheral tissues.
Peptides specific for melanocortin receptors have been reported to have a wide variety of biological activities, including effects upon pigmentation and steroidogenesis, known to be mediated by MSH (melanocyte stimulating hormone) and ACTH receptors. Several studies have documented the presence of melanotropin receptors on primary human melanoma cells (Tatro J B, Atkins M, Mier J W, et al. Melanotropin receptors demonstrated in situ in human melanoma. J Clin Invest, 85:1825–1832, 1990). Melanotropin receptors have been reported as markers for melanotic and amelanotic human melanoma tumors (Sharma S D, Granberry M E, Jiang J, et al. Multivalent melanotropic peptide and fluorescent macromolecular conjugates: new reagents for characterization of melanotropin receptors. J Clin Invest, 85:1825–1832, 1990). Melanotropin receptors have been reported as markers for melanotic and amelanotic human Sharma S D, Jiang J, Hadley M E, et al. Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors. Proc Natl Acad Sci USA 93(24):13715–13720, 1996). In particular, the presence of MC1-R has been demonstrated in human melanoma cells by an antibody to MC1-R (Xia Y, Skoog V, Muceniece R, et al. Polyclonal antibodies against human melanocortin MC-1 receptor: Preliminary immunohistochemical localization of melanocortin MC1 receptor to malignant melanoma cells. European J Pharmacol 288:277–283, 1995). MC1-R is a G protein-coupled, 7-transmembrane receptor expressed in skin-cell melanocytes and shares some degree of homology with related receptors MC2-R, MC3-R, MC4-R and MC5-R. Each of these receptors can bind various peptide analogs that contain a common melanotropic pharmacophore, His-Phe-Arg-Trp (SEQ ID NO:1), which describes the 6–9 sequence of the alpha-melanocyte stimulating hormone (α-MSH).
Prior to molecular characterization of the MC receptors, α-MSH analogs were labeled with the radioisotope Indium-111 and used in melanoma imaging studies (Wraight E P, Bard D R, Maughan T S, et al. The use of a chelating derivative of alpha melanocyte stimulating hormone for the clinical imaging of malignant melanoma. Brit J Radiology 65: 112–118, 1992; Bard D R, Knight C G and Page-Thomas D P. A chelating derivative of alpha-melanocyte stimulating hormone as a potential imaging agent for malignant melanoma. Brit J Cancer 62:919–922, 1990; Bard D R, Knight C G, Page-Thomas D P. Targeting of a chelating derivative of a short chain analogue of alpha-melanocyte stimulating hormone to Cloudman S91 melanomas. Biochem Soc Trans 18:882–883, 1990). Linear and cyclic disulfide-containing peptides have been identified and used for melanoma imaging and appear to be non-selective among MC receptors (Chen J and Quinn T P. Alpha melanocyte stimulating hormone analogues Tc-99 m/Re-188 labeling and their pharmacokinetics in malignant melanoma bearing mice. J Nucl Med 39: 222p, 1998; Giblin M F, Wang N, Hoffman T J, et al. Design and characterization of alpha-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination. Proc Natl Acad Sci USA 95(22):12814–12818, 1998). In later studies, the cyclic peptide reported by Giblin and coworkers was also found to localize in the brain (Wang N N, Giblin M F, Hoffman T J, et al. In vivo characterization of Tc-99m and Re-188 labeled cyclic melanotropin peptide analogues in a murine melanoma model. J Nucl Med 39: 77p, 1998 and corresponding poster presentation at the 45th Society of Nuclear Medicine Meeting, Toronto, June 1998). It has been recently reported that the response of human melanocytes to UV radiation is mediated by α-MSH induced activation of the cAMP pathway through the MC1-R (Im S, Moro O, Peng F, et al. Activation of the cyclic AMP pathway by alpha-melanotropin mediates the response of human melanocytes to ultraviolet B radiation. Cancer Res 58: 47–54, 1998).
MC4-R is also a G protein-coupled, 7-transmembrane receptor, but is believed to be expressed primarily in the brain. Inactivation of this receptor by gene targeting has been reported to result in mice with the maturity-onset obesity syndrome that is associated with hyperphagia, hyperinsulinemia, and hyperglycemia (Huszar D, Lynch C A, Fairchild-Huntress V, et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88:131–141, 1997). MC4-R is a molecular target for therapeutic intervention in energy homeostasis.
Alpha-MSH has been described as a potent anti-inflammatory agent in all major forms of inflammation (Star R A, Rajora N, Huang J, Stock R C, Catania A, and Lipton J M: Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte stimulating hormone. Proc Natl Acad Sci USA 92:8016–8020, 1995; Getting S J, and Perretti M: MC3-R as a novel target for antiinflammatory therapy. Drug News and Perspectives 13:19–27, 2000). Implication of both MC1-R and MC3-R receptors in anti-inflammatory processes has been stressed. In particular, the activation of these MC receptors by melanocortin receptor agonists has been reported to inhibit the expression of nitric oxide synthase and subsequent nitric oxide production.
Significant work has been done in determining the structure of melanocortin receptors, including both the nucleic acid sequences encoding for the receptors and the amino acid sequences constituting the receptors. See, for example, International Patent Applications No. PCT/US98/12098 and PCT/US99/16862 and U.S. Pat. No. 5,994,087. A large number of ligands specific for melanocortin receptors, both agonists and antagonists, have also been developed. See, for example, International Patent Applications No. PCT/US98/03298 (iodo group-containing melanocortin receptor-specific linear peptide), PCT/GB99/01388 (MC1-R specific linear peptides), PCT/GB99101195 (MC3-R, MC4-R and MC5-R specific cyclic peptides), PCT/US99/04111 (MC1-R specific peptide antagonists for melanoma therapy), PCT/US99/09216 (isoquinoline compounds as melanocortin receptor ligands), PCT/US99/13252 (spiropiperdine derivatives as melanocortin receptor agonists), and U.S. Pat. No. 6,054,556 (cyclic lactam peptides as MC1-R, MC3-R, MC4-R and MC5-R antagonists). In addition, a large number of patents teach various methods of screening and determining melanocortin receptor-specific compounds, as for example International Patent Applications No. PCT/US97115565, PCT/US98112098 and PCT/US99/16862 and U.S. Pat. Nos. 5,932,779 and 5,994,087.
In general, compounds specific for MC1-R ar believed to be useful for treatment of melanoma, including use as radiotherapeutic or drug delivery agent, and as diagnostic imaging agents, particularly when labeled with a diagnostic radionuclide. Compounds specific for MC3-R, MC4-R or MC5-R are believed to be useful in regulation of energy homeostasis, including use as agents for attenuating food intake and body weight gain, for use in treatment of anorexia, as a weight gain aid, for treatment of obesity, and other treatment of other food intake and metabolism-related purposes. Compounds specific for MC3-R and MC4-R, among other melanocortin receptors, can be used as agents for treatment of sexual dysfunction, including male erectile dysfunction. Compounds specific for MC3-R and MC4-R, among other melanocortin receptors, can be used to regulate blood pressure, heart rate and other neurophysiologic parameters. Other melanocortin receptor peptides can be used as tanning agents, to increase melanin production, such as peptides that are MCR-1 agonists. Compounds specific for MCR-1 and MCR-3 may be useful in regulation of inflammatory processes.
There remains a significant need for ligands with high specificity for discrete melanocortin receptors, as well as ligands or compounds that are either agonists or antagonists of specific melanocortin receptors. High affinity peptide ligands of melanocortin receptors can be used to exploit varied physiological responses associated with the melanocortin receptors, either as agonists or antagonists. In addition, melanocortin receptors have an effect on the activity of various cytokines, and high affinity peptide ligands of melanocortin receptors can be used to regulate cytokine activity.
Peptide Libraries and Combinatorial Chemistry. Libraries of peptides and other small molecules, with enormous pools of structurally diverse molecules, are well suited for pharmaceutical lead generation and lead optimization. Libraries of a variety of molecular species have been described in literature and screened for drug discovery, including peptides, peptoids, peptidomimetics, oligonucleotides, benzodiazepines, and other libraries of small organic molecules. Various approaches have been used to construct libraries of structurally diverse chemical compounds, include including chemical synthesis and genetic engineering methods. Chemically synthesized libraries have been synthesized by general solution chemical means and by solid-phase methods. The prior art on designing, synthesizing, screening, and evaluation of peptide-based libraries has been reviewed in numerous articles.
Spatially Addressable Parallel Synthesis of Solid Phase Bound Libraries. Various strategies for chemical construction of a library of peptides or other small molecules are well established. One strategy involves spatially separate synthesis of compounds in parallel on solid phase or on a solid surface in a predetermined fashion so that the location of one compound or a subset of compounds on the solid surface is known. Other methods, such as light-directed spatially addressable parallel chemical synthesis techniques, based upon use of photolithographic techniques in peptide synthesis on a solid surface, such as a borosilicate glass microscope slide, provide libraries containing more than 100,000 spatially separated compounds. How ver, synthesis of libraries that are structurally more diverse than simple peptides requires the development of orthogonal photolabile protecting groups that can be cleaved at different wavelengths of light. In addition, the solid surface bearing these libraries also has been reported to cause a pronounced effect on binding affinities in library screening assays.
Pooling and Split Synthesis Strategies. Large libraries of compounds can be assembled by a pooling strategy that employs equimolar mixtures of reactants in each synthetic step or by adjusting the relative concentration of various reactants in the mixture according to their reactivities in each of the coupling reactions. In one approach equimolar mixtures of compounds are obtained by splitting the resin in equal portions, each of which is separately reacted with each of the various monomeric reagents. The resin is mixed, processed for the next coupling, and again split into equal portions for separate reaction with individual reagents. The process is repeated as required to obtain a library of desired oligomeric length and size. This approach is the basis of the “one-bead one-peptide” strategy which employs amino acid sequencing to ascertain the primary structure of the peptide on a hit bead in a bioassay. Automated systems have been developed for carrying out split synthesis of these libraries with rather more efficiency. A common artifact occasionally seen with all these resin bound libraries is altered target-specific affinity by some solid phase bound compounds in bioassays, which can result in totally misleading results.
Another strategy involves construction of soluble libraries. This strategy involves a deconvolution process of iterative re-synthesis and bioassaying until all the initially randomized amino acid positions are defined. Several modifications to this strategy have been developed, including co-synthesis of two libraries containing orthogonal pools, which eliminates the need of iterative re-synthesis and evaluation. A major limitation of the soluble library approach is its applicability to high affinity systems. The abundance of each compound in solution can be influenced by the total number of compounds in a library that can affect the biological activity. For this reason, a highly active compound in any pool may not in fact be the most potent molecule. Lack of reasonable solubilities of certain members in a library may further influence this phenomenon.
Among the various classes of libraries of small molecules, peptide libraries remain the most versatile because of the structural diversity offered by the use of naturally occurring amino acids, incorporation of a variety of “designer” amino acids, and the high efficiency and ease with which peptide synthesis can be accomplished. In addition, another level of structural diversity in peptide-based libraries has been added by post-synthesis modification of the libraries. These modifications include permethylation, acylation, functionalization of the side chain functionality, and reductive amination of the N-terminus.